Microscopes, beam splitters and microscope systems

Abstract

A microscope apparatus includes: an opening (31) having a first slit and a second slit through which multiple beams of light pass, the multiple beams of light originating from an object of observation and generated by multiple beams of illumination light having different wavelengths emitted to the object of observation; a dispersive element for dispersing the wavelength of each of the multiple beams of light passing through the opening (31); and an imaging element (32) for receiving the multiple beams of light whose wavelengths have been dispersed by the dispersive element. The imaging element (32) receives the multiple beams of light dispersed by wavelength such that the zero-order light passing through the second slit and the first-order light passing through the first slit do not overlap.

Description

Technical Field

[0001] The present disclosure relates to a microscope apparatus, a beam splitter and a microscope system. Background Art

[0002] Generally, regarding the technology for acquiring imaging data of an observation object, for example, there is fluorescence microscopy for pathological image acquisition or imaging cytometry. As a method excellent in quantitative and multicolor analysis, various image diagnosis methods using fluorescent staining have been proposed.

[0003] Because multiplexing is easier and because detailed diagnostic information can be obtained, fluorescence microscopy is more advantageous than the staining performed by coloring. In fluorescence imaging except for the fluorescence imaging for pathological diagnosis, the increase in the number of colors makes it possible to once check the various antigens occurring in the sample. In general fluorescence imaging, excitation light is emitted with the absorption wavelength (excitation wavelength) of dye, and the dye spectrum generated from the emitted light is selectively captured using a bandpass filter. In the presence of multiple colors, the absorption wavelength (excitation wavelength) changes according to the dye. Therefore, the method of capturing by changing the filter for each dye is adopted. However, the absorption spectrum and emission spectrum of the dye are wide and overlap each other. So, in the case of using multiple colors to dye, multiple dyes are excited at an excitation wavelength. In addition, the fluorescence of adjacent dyes overflows into the bandpass filter, and color mixing (overlap of fluorescence) occurs.

[0004] On the other hand, a method of photographing by changing the wavelength of the excitation light and the wavelength of the fluorescence to be detected in a time-division manner is known. However, this method has a problem that the photographing time increases linearly as the number of colors increases. As an observation method taking the above into consideration, there is a fluorescence microscope using multiple beams of excitation light and multiple slits. According to this method, multiple beams of excitation light can be emitted at a time, and the data of the fluorescence generated by all the excitations can be obtained by a single scan.

[0005] Prior art literature

[0006] Patent Document 1: Japanese Patent Publication No. 4452850

[0007] Non-patent literature 1: Edward C. Stack, "Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis", Methods 70 (2014) 46-58 Summary of the invention

[0008] Problems to be solved by the present invention

[0009] However, the above-mentioned conventional fluorescence microscope using multiple excitation lights and multiple slits has the following problems: for example, in the imaging unit, the first-order light of light passing through one slit and the zero-order light of light passing through other slits overlap each other, and accurate imaging data cannot be obtained.

[0010] Therefore, the present disclosure has been made in view of the above situation, and a microscope device, a spectrometer and a microscope system are proposed, which are capable of obtaining accurate imaging data through an imaging unit by illuminating an observation object with multiple illumination lights with different wavelengths and allowing multiple beams of light from the observation object to pass through multiple slits and then undergo wavelength dispersion.

[0011] Solution to the problem

[0012] In order to solve the above-mentioned problem, a microscope device according to the present disclosure includes: an opening portion including a first slit and a second slit, through which a plurality of beams of light from an observation object pass, the plurality of beams of light being generated by emitting a plurality of irradiation lights having different wavelengths to the observation object; a dispersion element for performing wavelength dispersion on the plurality of beams of light passing through the opening portion; and an imaging element for receiving the plurality of beams of light wavelength-dispersed by the dispersion element. The imaging element performs light reception so that, for the wavelength-dispersed plurality of beams of light, zero-order light of the light passing through the second slit and first-order light of the light passing through the first slit do not overlap each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a schematic block diagram showing a fluorescence observation apparatus according to an embodiment.

[0014] Figure 2 : is a diagram showing an example of an optical system in the fluorescence observation apparatus according to the present embodiment.

[0015] Figure 3 This is a diagram for explaining light scattering by a diffraction grating.

[0016] Figure 4 is a schematic diagram of a pathological sample serving as an observation object according to the present embodiment.

[0017] Figure 5 Schematic diagram showing a state of linear illumination light emitted toward an observation object according to the present embodiment.

[0018] Figure 6 yes Figure 2 An enlarged view of the spectral imaging unit 30 in FIG.

[0019] Figure 7: is a graph showing the positions of the zero-order light and the first-order light of the slit #1 and the zero-order light and the first-order light of the slit #2 in the imaging unit in the present embodiment.

[0020] Figure 8 : is a graph showing a first example in this embodiment.

[0021] Fig. 9 is a schematic diagram showing the field of view of imaging data provided by the imaging unit in this embodiment.

[0022] Fig.10 : is a graph showing a second example in this embodiment.

[0023] Fig.11 is a graph showing a simulation example of the diffraction efficiency in this embodiment.

[0024] Fig.12 : is a diagram showing a third example in this embodiment.

[0025] Fig.13 : is a graph showing a fourth example in this embodiment.

[0026] Fig.14 : is a graph showing a fifth example in this embodiment.

[0027] Fig.15 : is a graph showing a sixth example in this embodiment.

[0028] Fig.16 : is a graph showing a seventh example in this embodiment.

[0029] Fig.17 : is a graph for describing the limitation of the wavelength range to be observed in this embodiment.

[0030] Fig.18 : is a graph for describing data correction of second-order light in this embodiment.

[0031] Fig.19 : is a graph for describing a specific example of data correction of second-order light in this embodiment.

[0032] Fig. 20 : is a graph for describing data correction of negative first-order light in this embodiment.

[0033] Fig.21 : is a diagram for describing a specific example of data correction in this embodiment. DETAILED DESCRIPTION

[0034] Hereinafter, an embodiment of the present disclosure will be described in detail with reference to the accompanying drawings. In this embodiment, a fluorescence observation device that observes fluorescence from an observation target will be described as an example of a microscope device.

[0035] First, refer to Figure 1 and Figure 2 The configuration of the fluorescence observation device 100 according to the present embodiment will be described. Figure 1 is a schematic block diagram showing the fluorescence observation apparatus 100 according to the present embodiment. Figure 2 2 is a diagram showing an example of the optical system of the fluorescence observation apparatus 100 according to the present embodiment.

[0036] The fluorescence observation device 100 of the present embodiment has an observation unit 1. The observation unit 1 has an excitation unit 10, a stage 20, and a spectral imaging unit 30 (hereinafter also referred to as a spectroscope). The excitation unit 10 (irradiation unit) irradiates an observation target with a plurality of irradiation lights having different wavelengths. For example, the excitation unit 10 irradiates a pathological sample (pathological sample) serving as an observation target with a plurality of linear illumination lights having different wavelengths arranged in a parallel and spatially separated state.

[0037] Here, the state of parallel and spatial separation refers to a state in which a plurality of linear illumination lights have different axes and are parallel. Having different axes means not being on the same axis, and the distance between these axes is not particularly limited. Parallelism is not limited to parallelism in a strict sense, and includes a state of being substantially parallel. For example, there may be distortion caused by an optical system such as a lens or a deviation from a parallel state due to manufacturing tolerance, and this case is also considered to be parallel.

[0038] The stage 20 is a base for supporting the pathological sample. The spectral imaging unit 30 acquires the fluorescence spectrum (spectral data) of the pathological sample excited in a line form.

[0039] The fluorescence observation device 100 also includes a processing unit 2. The processing unit 2 generally forms an image of the pathological sample or outputs a distribution of the fluorescence spectrum based on the fluorescence spectrum of the pathological sample (hereinafter, also referred to as the sample S) acquired by the observation unit 1. The image mentioned herein refers to the dye constituting the spectrum, the component ratio of the autofluorescence derived from the sample, etc., the RGB (red, green, and blue) color converted from the waveform, the brightness distribution in a specific wavelength band, etc.

[0040] The excitation unit 10 and the spectral imaging unit 30 are connected to the stage 20 via an observation optical system 40 such as an objective lens 44. The observation optical system 40 has a function of maintaining optimal focus through a focusing mechanism 60. In addition, a non-fluorescence observation unit 70 for performing dark field observation, bright field observation, etc. may be connected to the observation optical system 40.

[0041] Furthermore, the fluorescence observation apparatus 100 may be connected to a control unit 80 that controls the excitation unit 10 , the spectral imaging unit 30 , the scanning mechanism 50 , the focusing mechanism 60 , the non-fluorescence observation unit 70 , and the like.

[0042] The excitation unit 10 includes a plurality of light sources L1, L2, ... that can output light having a plurality of excitation wavelengths Ex1, Ex2, .... Each of the plurality of light sources generally includes a light emitting diode (LED), a laser diode (LD), a mercury lamp, etc., and each light is irradiated and emitted to a sample S on a stage 20 in a line form.

[0043] Here, Figure 4 Schematic diagram of a pathological sample used as an observation object according to the present embodiment. Figure 5 Schematic diagram showing a state where a linear illumination light is irradiated to an observation object in this embodiment. The sample S usually includes a slide glass, which includes the observation object Sa, for example, Figure 4 The tissue slice shown in FIG. 1 is a tissue slice, but may be in another form. The observation target Sa is a biological sample such as nucleic acid, cells, proteins, bacteria, and viruses. The sample S (observation target Sa) is stained with a plurality of fluorophores. The observation unit 1 magnifies and observes the sample S at a desired magnification. When the magnification is Figure 4 When part A in Figure 5 As shown in , a plurality of linear illumination lights (two (Ex1 and Ex2) in the example shown) are arranged in the illumination area. The shooting areas R1 and R2 of the spectral imaging unit 30 are arranged to overlap with the corresponding illumination areas. The two linear illumination lights Ex1 and Ex2 are emitted in parallel in the Z-axis direction and are arranged to be separated from each other by a predetermined distance (Δy) in the Y-axis direction.

[0044] Furthermore, the spectral imaging unit 30 includes an opening portion 31 , an imaging element 32 , a prism 33 , a reflection mirror 34 , and a diffraction grating 35 . Figure 2 The opening 31 shown has a plurality of slits, through which the plurality of beams of light from the observation object generated by the plurality of illumination lights pass. The imaging regions R1 and R2 correspond to the slits of the opening 31. That is, the slits of the spectral imaging unit 30 are configured in the same number as the number of linear illumination lights.

[0045] The wavelength of the first linear illumination light Ex1 and the wavelength of the second linear illumination light Ex2 are different from each other. The linear fluorescence excited by the linear illumination lights Ex1 and Ex2 is observed in the spectral imaging unit 30 via the observation optical system 40.

[0046] The imaging element 32 (imaging unit) images (receives) multiple beams of light (fluorescence, etc.) that are wavelength-dispersed by the diffraction grating 35 (spectrometric element). As the imaging element 32, a two-dimensional imager such as a charge coupled device (CCD) imager and a complementary metal oxide semiconductor (CMOS) imager is used.

[0047] The spectral imaging unit 30 uses the pixel array in one direction (for example, the vertical direction) of the imaging element 32 as a channel of wavelength to acquire the spectral data (x, λ) of the fluorescence from each of the row illumination lights Ex1 and Ex2. The acquired spectral data (x, λ) is recorded in the processing unit 2 in a state that it is clear at which excitation wavelength the spectral data is acquired by excitation.

[0048] The processing unit 2 (correction unit) processes the imaging data received from the imaging element 32. For example, the processing unit 2 is implemented by hardware elements used in a computer, such as a central processing unit (CPU), a random access memory (RAM), and a read-only memory (ROM), and a required program (software). Instead of or in addition to the CPU, a programmable logic device (PLD) such as a field programmable gate array (FPGA), a digital signal processor (DSP), an application specific integrated circuit (ASIC), etc. may be used.

[0049] The processing unit 2 includes a storage unit 21. The storage unit 21 stores spectral data representing the correlation between the wavelengths of the multiple beams of linear illumination light Ex1, Ex2 and the fluorescence received by the imaging element 32. As the storage unit 21, a storage device such as a non-volatile semiconductor memory and a hard disk drive is used, and the storage unit 21 has a standard spectrum for the autofluorescence of the sample S and a standard spectrum of the dye itself for dyeing the sample S stored therein. The spectral data (x, λ) received by the imaging element 32 is stored in the storage unit 21. In an embodiment, a storage unit storing the standard spectrum of the autofluorescence of the sample S and the dye itself and a storage unit storing the spectral data (measured spectrum) of the sample S acquired by the imaging element 32 are composed of a common storage unit 21, but the present disclosure is not limited thereto, and they may be composed of separate storage units. In addition, with respect to the light information obtained from the multiple wavelength dispersed lights respectively, the processing unit 2 can correct the light information obtained from the light passing through the first slit based on the light information obtained from the light passing through the second slit (the details will be described below).

[0050] In addition, if Figure 2 As shown, a dichroic mirror 42 and a bandpass filter 45 are inserted in the middle of the optical path so that the excitation light ( Ex1 and Ex2 ) cannot reach the imaging element 32 .

[0051] The diffraction grating 35 is arranged on the optical path between the opening 31 and the imaging element 32. The diffraction grating 35 is an example of a wavelength dispersion element that performs wavelength dispersion on the multiple beams of light that have passed through the multiple slits of the opening 31. The diffraction grating 35 causes wavelength dispersion due to the coherence of the light. Generally, the diffraction grating has the following advantages: the dependence of the wavelength dispersion on the wavelength is basically fixed, and the wavelength dispersion is easy to design based on the grating pitch. That is, by using the diffraction grating 35, it is possible to easily design how to generate wavelength dispersion when light is incident on the imaging element 32.

[0052] In addition, the diffraction grating 35 is, for example, a blazed diffraction grating (blazed grating) in which the cross-sectional shape of the grooves is sawtooth-shaped. By using a blazed diffraction grating, a large amount of light of a specific order can be obtained. For example, the wavelength component of the second-order light can be suppressed. Therefore, even if the second-order light of other slits overlaps with the first-order light of a specific slit, the influence of color mixing can be suppressed to be small, and the spectrum of each slit can be accurately obtained. In addition, by using a blazed diffraction grating, negative first-order light can be suppressed to be smaller than first-order light. Therefore, for example, even if the first-order light of a specific slit overlaps with the negative first-order light of other slit #2, the influence of the negative first-order light can be reduced.

[0053] here, Figure 3 is an explanatory diagram of light dispersion of a diffraction grating. When a diffraction grating is used, for example, negative third-order light to third-order light are generated in response to incident light (wavelength λ1 to λ2). Hereinafter, the problems of the conventional technology will be described in detail.

[0054] In a fluorescence microscope using multiple beams of excitation light and multiple slits, it is necessary to use a spectroscope to split the excitation light and use first-order light or negative first-order light to perform observation. However, in reality, undispersed zero-order light and undesired high-order light (second-order light or above) are generated. Then, since each beam of light passing through the multiple slits is dispersed, the diffracted light obtained from the other slits overlaps with the area of ​​the diffracted light obtained from the specific slit in the imaging unit, and an accurate spectrum cannot be obtained. In the past, in many spectroscopes, one slit was formed for one spectroscopic element, and therefore, this problem was not obvious.

[0055] For example, the case where there are two slits will be considered as an example. Ideally, images of two scattered first-order lights (or negative first-order lights) will be projected on the imager (imaging element). However, in reality, zero-order light and high-order light are generated, so it is not always possible to acquire only the ideal first-order light image. For example, the zero-order light of the light from slit #1 may overlap with the first-order light (or negative first-order light) of the light from slit #2, or the high-order light (e.g., second-order light) of the light from slit #1 and the first-order light (or negative first-order light) of the light from slit #2 may overlap with each other. In the case where such an overlap occurs in this way, the spectrum of the first-order light (or negative first-order light) to be observed cannot be accurately measured.

[0056] In this case, in this embodiment, a method will be described in which the imaging unit can acquire accurate (more accurate than conventional techniques) imaging data by avoiding or reducing such overlap.

[0057] return Figure 1 and Figure 2 , the observation unit 1 further includes a scanning mechanism 50. The scanning mechanism 50 changes the position of the observation object irradiated with the irradiation light over time. For example, the scanning mechanism 50 can scan the multiple beams of linear illumination light Ex1 and Ex2 along the Y-axis direction (i.e., the arrangement direction of the linear illumination light Ex1 and Ex2) on the stage 20. By driving the scanning mechanism 50, the imaging element 32 continuously images the multiple beams of wavelength-dispersed light in the scanning direction over time, and the processing unit 2 processes the imaging data received from the imaging element 32 to obtain two-dimensional data or three-dimensional data of the observation object.

[0058] Specifically, by using the scanning mechanism 50 , dye spectra (fluorescence spectra) spatially separated by Δy on the sample S (observation target Sa) and excited at different excitation wavelengths can be continuously recorded in the Y-axis direction.

[0059] Note that in the above example, although the number of linear illumination lights used as excitation lights is two, the number of linear illumination lights is not limited to two, and may be three, four, or five or more. In addition, each linear illumination light may include a plurality of excitation wavelengths selected so that the color separation performance is not degraded as much as possible.

[0060] Next, refer to Figure 2 The details of the observation unit 1 are described. The excitation unit 10 includes a plurality of (four in this example) excitation light sources L1, L2, L3, and L4. The excitation light sources L1 to L4 include laser sources that output laser light having wavelengths of 405 nm, 488 nm, 561 nm, and 645 nm, respectively.

[0061] The excitation unit 10 further includes a plurality of collimating lenses 11 , a laser line filter 12 , dichroic mirrors 13 a , 13 b , and 13 c , a homogenizer 14 , a condenser lens 15 , and an incident slit 16 so as to correspond to the respective excitation light sources L1 to L4 .

[0062] The laser light emitted from the excitation light source L1 and the laser light emitted from the excitation light source L3 are collimated by the collimating lens 11, pass through the laser line filter 12 for cutting the edge of each wavelength band, and are made coaxial by the dichroic mirror 13a. The two coaxial laser beams are further beam-shaped by the homogenizer 14 (such as a fly-eye lens) and the condenser lens 15 to become the linear illumination light Ex1.

[0063] Similarly, the laser emitted from the excitation light source L2 and the laser emitted from the excitation light source L4 are made coaxial by dichroic mirrors 13b and 13c, and become linear illumination light Ex2 having an axis different from the axis of the linear illumination light Ex1. The linear illumination light beams Ex1 and Ex2 form different axis linear illumination lights (original images) separated by Δy in the incident slit 16 (conjugate slit), which has a plurality of slits through which the linear illumination light beams Ex1 and Ex2 can pass. Note that in the present embodiment, an example in which four lasers are made into two coaxial lasers and two non-coaxial lasers is described, but alternatively, the two lasers may be made into two non-coaxial lasers, or the four lasers may be made into four non-coaxial lasers.

[0064] The primary image is emitted to the sample S on the stage 20 through the observation optical system 40. The observation optical system 40 includes a condenser lens 41, dichroic mirrors 42 and 43, an objective lens 44, a bandpass filter 45, and a condenser lens 46 (imaging lens). The linear illumination light sheets Ex1 and Ex2 are collimated by the condenser lens 41 paired with the objective lens 44, reflected by the dichroic mirrors 42 and 43, pass through the objective lens 44, and emitted to the sample S.

[0065] like Figure 5 The illumination lights shown in FIG. 4 are formed on the surface of the sample S. Fluorescence excited by these illumination lights is condensed by the objective lens 44 , reflected by the dichroic mirror 43 , passes through the dichroic mirror 42 and the bandpass filter 45 that cuts off the excitation light, condensed again by the condensing lens 46 , and incident on the spectral imaging unit 30 .

[0066] The opening 31 is arranged at the focal point of the condenser lens 46, and has the same number of slits as the number of excitation lines. The fluorescence spectrum derived from the two excitation lines passing through the opening 31 is reflected by the prism 33 and the reflection mirror 34, then wavelength-separated by the diffraction grating 35, reflected again by the reflection mirror 34, and then incident on the imaging element 32.

[0067] The stage 20 and the scanning mechanism 50 constitute an XY stage, and move the sample S in the direction of the X axis and the direction of the Y axis to acquire a fluorescent image of the sample S. In whole slide imaging (WSI), the operation of scanning the sample S in the direction of the Y axis, then moving in the direction of the X axis, and further scanning the sample S in the direction of the Y axis is repeated.

[0068] The non-fluorescence observation unit 70 includes a light source 71 , a dichroic mirror 43 , an objective lens 44 , a condenser lens 72 , an imaging element 73 , and the like. Figure 2 A non-fluorescence observation system is an observation system that uses dark field illumination.

[0069] The light source 71 is arranged below the stage 20, and illuminates the sample S on the stage 20 with illumination light from the opposite side of the linear illumination lights Ex1 and Ex2. In the case of dark field illumination, the light source 71 illuminates the sample S from outside the numerical aperture (NA) of the objective lens 44, and the light (dark field image) diffracted by the sample S is captured by the imaging element 73 via the objective lens 44, the dichroic mirror 43, and the condenser lens 72. By using dark field illumination, even apparently transparent samples (such as fluorescently labeled samples) can be observed with contrast.

[0070] Note that the dark field image can be observed simultaneously with the fluorescence and used for real-time focusing. In this case, a wavelength that does not affect the fluorescence observation can be selected as the illumination wavelength. The non-fluorescence observation unit 70 is not limited to an observation system that acquires a dark field image, and may include an observation system that can acquire non-fluorescence images such as bright field images, phase difference images, phase images, and online holographic images. For example, as a method for acquiring a non-fluorescence image, various observation methods such as the Schlieren method, the phase difference contrast method, the polarization observation method, and the epi-illumination method can be used. The position of the illumination light source is not limited to the position below the stage, and the light source may be located above the stage or around the objective lens. In addition, not only a method for performing focus control in real time can be used, but also other methods such as a pre-focus mapping method that pre-records the focus coordinates (Z coordinates) can be used.

[0071] The fluorescence spectrum acquired by the imaging element 32 is output to the processing unit 2. The processing unit 2 includes a data calibration unit 22 and an image forming unit 23 in addition to the storage unit 21. The data calibration unit 22 corrects the spectrum data stored in the storage unit 21. The image forming unit 23 forms a fluorescence image of the sample S based on the spectrum data and the interval Δy between the plurality of linear illumination lights Ex1 and Ex2.

[0072] The display unit 3 displays various information generated by the processing unit 2. The display unit 3 may include, for example, a monitor integrally attached to the processing unit 2, or may be a display device connected to the processing unit 2. The display unit 3 includes, for example, a display element such as a liquid crystal device and an organic EL device, and a touch sensor, and functions as a user interface (UI) that allows input and setting of shooting conditions and displays a shot image and the like.

[0073] The following describes in detail the structure of the observation unit 1 that obtains accurate imaging data using the imaging element 32. The observation unit 1 is configured such that, for the plurality of wavelength-dispersed lights, the zero-order light of the specific light in the imaging element 32 does not overlap with the first-order light of the other lights. In addition, it is preferred that the wavelength band of the first-order light of at least one wavelength-dispersed light among the plurality of wavelength-dispersed lights is part or all of 400nm to 800nm. The reason for this is that the wavelength of the fluorescence mainly used for fluorescence observation of a living body is 400nm to 800nm. However, the wavelength band is not limited thereto.

[0074] Figure 6 yes Figure 2 Here, an example of the case where the Offner type is adopted as the spectral imaging unit 30 will be described. In addition, Figure 7 : is a graph showing the positions of the zero-order light and the first-order light of the slit #1 (first slit) and the zero-order light and the first-order light of the slit #2 (second slit) in the imaging unit (imaging element 32) in the present embodiment. The horizontal axis represents the Y coordinate position of the imaging element 32, and the vertical axis represents the light intensity (I). It should be noted that, for example, the zero-order light of the light passing through the slit #1 is referred to as the "zero-order light of the slit #1", and the same applies to other light.

[0075] The reflector 34 is a concave reflector having a reflective surface with a curvature radius of R on the inner side. In addition, the diffraction grating 35 is a reflective diffraction grating (convex mirror) having a reflective surface with a curvature radius of R / 2 on the outer side. Furthermore, two slits (slit #1 and slit #2) are provided in the opening 31. The distance between the slit #1 and the slit #2 is referred to as L.

[0076] The y-coordinate position of the zero-order light of the slit #1 in the imaging element 32 is referred to as y1. The y-coordinate position of the zero-order light of the slit #2 in the imaging element 32 is referred to as y2.

[0077] The y coordinate position of the first-order light of slit #1 in the imaging element 32 is referred to as y11 to y12, and its wavelength band is referred to as λ min1 To max1 The y coordinate position of the first-order light of slit #2 in the imaging element 32 is referred to as y21 to y22, and its wavelength band is referred to as λ min2 To max2 . The diffraction order is called m.

[0078] In this case, y11, y12, y21, and y22 are expressed as equations (1) to (4) provided below.

[0079]

[0080]

[0081]

[0082]

[0083] Furthermore, considering the first-order light, since the diffraction order m=1 is established, y11, y12, y21, and y22 are expressed as equations (5) to (8) provided below.

[0084]

[0085]

[0086]

[0087]

[0088] here, Figure 8 y2-y1 is a diagram showing a first example in the present embodiment. In the first example, the observation unit 1 is configured so that the zero-order light of slit #2 is located between the zero-order light of slit #1 and the first-order light of slit #1 in the imaging element 32. Furthermore, L=y2-y1 is established.

[0089] The conditions are shown in the formula (9) provided below.

[0090]

[0091] When y1 is subtracted from each term of equation (9), equation (10) provided below is obtained, which is also the condition for the slit distance L.

[0092]

[0093] As described above, according to the first example, for slits #1 and #2, since the zero-order light of one slit does not overlap with the first-order light of the other slits in the imaging element 32, accurate imaging data can be obtained in the imaging element 32. That is, more accurate diffraction data of the first-order light (with less noise) can be obtained.

[0094] Here, Fig. 9 is a schematic diagram showing the field of view of the imaging data provided by the imaging unit (imaging element 32) of the present embodiment. In the present embodiment, the slit distance is equal to or less than the field of view diameter of the imaging data provided by the imaging element 32, which is defined by the objective lens 44 and the focusing lens 46. In addition, the first example has the following configuration: In the second to seventh examples below, the slit distance between slits #1 and #2 can be the shortest. The imaging performance is better in the area closer to the center in the field of view. Since the slit distance can be shorter, the imaging performance can be further improved.

[0095] Specifically, in fluorescence observation of organisms, fluorophores having fluorescence peaks of about 400 nm to 800 nm are often used (for example, fluorophore Indo-1 is fluorescent at about 400 nm, and APC Alexa Fluor 750 is fluorescent at about 800 nm). Therefore, when the wavelength band of the first-order light of at least one wavelength-dispersed light among a plurality of wavelength-dispersed lights is a part or all of 400 to 800 nm, the accuracy of fluorescence observation of the organism can be improved.

[0096] Note that although a configuration using first-order light is illustrated, the same applies to a case where negative first-order light is used.

[0097] Next, a second example will be described. Fig.10 : is a diagram showing a second example in the embodiment. The second example is an example in which, in the imaging element 32, the observation unit 1 is configured so that the zero-order light of the slit #2 is located on the second-order light side with respect to the first-order light of the slit #1.

[0098] The conditions are shown in the formula (11) provided below.

[0099]

[0100] When y1 is subtracted from each term of equation (11) and the diameter of the observation field is D, the condition of the slit distance L (= y2 - y1) is expressed by the following equation (12).

[0101]

[0102] As described above, according to the second example, regarding slits #1 and #2, since the zero-order light of one slit does not overlap with the first-order light of the other slits in the imaging element 32, accurate imaging data can be obtained in the imaging element 32. That is, more accurate diffraction data of the first-order light (with less noise) can be obtained.

[0103] In addition, as described above, by using a blazed diffraction grating as the diffraction grating 35, a large amount of light of a specific order can be obtained. For example, the wavelength component of the second-order light can be suppressed. Therefore, for example, a part of the second-order light of slit #1 overlaps with the first-order light of slit #2, but the influence can be small.

[0104] Here, Fig.11 is a graph showing a simulation example of the diffraction efficiency in this embodiment. Obviously, the diffraction efficiency of the first-order light is high, and the diffraction efficiency of the second-order light and the diffraction efficiency of the third-order light are low. Note that although the diffraction efficiency of the second-order light is slightly higher on the short wavelength side, the influence is small because, as Fig.10As shown, the portion where the second-order light from slit #1 overlaps with the first-order light from slit #2 is on the long wavelength side. Therefore, the influence of color mixing can be small, and an accurate spectrum can be obtained. In addition, in the case of using a blazed diffraction grating, the diffraction efficiency of the negative first-order light relative to the negative third-order light can be made small enough to be ignored.

[0105] Furthermore, the second example has a configuration in which, although the slit distance between slits #1 and #2 is not as short as in the first example, the slit distance between slits #1 and #2 can be shorter than in the third to seventh examples below, and high image forming performance can be achieved.

[0106] Hereinafter, third to seventh examples will be described, but since they are similar to those in the first or second example, description of the formulas will be omitted.

[0107] A third example will be described. Fig.12 is a graph showing a third example in this embodiment. Compared with the second example, the observation unit 1 is configured such that the second-order light of slit #1 does not overlap with the first-order light of slit #2, and the third-order light of slit #1 overlaps with the first-order light of slit #2.

[0108] Generally, the lower the diffraction efficiency of the diffracted light, the higher the order. Therefore, since it is not the second-order light of slit #1 but the third-order light of slit #1 that overlaps with the first-order light of slit #2, the accuracy of the imaging data can be further improved. Note that the slit distance can be determined within a range that can ensure sufficient image formation performance.

[0109] Next, a fourth example will be described. Fig.13 is a diagram showing a fourth example in this embodiment. Fig.10 Here, a method of suppressing the influence of high-order light mixing between the slits by data correction is considered. For example, the wavelength and intensity of high-order light overlapping with the first-order light of slit #1 (region A) and the first-order light of slit #2 (region B) are pre-evaluated (calculated). By correcting the data of the color mixing portion based on this evaluation, an accurate spectrum can be obtained even in the case where color mixing occurs.

[0110] Although color mixing between multiple slits has been described here, second-order light and first-order light may overlap each other in one slit. This can be handled by evaluating the overlapping portion in advance and performing data correction similarly to the above method, or by limiting the wavelength band reaching the imaging element 32 using an optical filter for fluorescence from the observation object. These can be combined.

[0111] Next, a fifth example will be described. Fig.14: is a diagram showing a fifth example in the present embodiment. A case where the number of slits is two has been described above, but the number of slits may be three or more. Fig.14 The example in shows the case where the number of slits is three in the first example. The first-order light of slit #1 is arranged between the zero-order light of slit #2 and the zero-order light of slit #3.

[0112] Note that the distances of the three first-order lights are not necessarily the same and can be set to be different. Therefore, the slit distances are not necessarily the same and can be different.

[0113] As described above, according to the fifth example, even when there are three slits, the zero-order light of a specific slit does not overlap with the first-order light of other slits, so accurate imaging data can be obtained in the imaging element 32. That is, more accurate diffraction data of the first-order light (with less noise) can be obtained.

[0114] Next, a sixth example will be described. Fig.15 : is a diagram showing a sixth example in the embodiment. Fig.15 The example in shows the case where the number of slits is four in the first example. The first-order light of slit #2 is arranged between the zero-order light of slit #3 and the zero-order light of slit #4.

[0115] As described above, according to the sixth example, even when the number of slits is 4, since the zero-order light of a specific slit does not overlap with the first-order light of other slits, accurate imaging data can be obtained in the imaging element 32. That is, more accurate diffraction data of the first-order light (with less noise) can be obtained. Note that the same can be achieved when the number of slits is five or more.

[0116] Next, a seventh example will be described. Fig.16 is a graph showing a seventh example in the embodiment. Fig.16 In the example of , the zero-order light of slit #2 is arranged in the area of ​​the second-order light of slit #1, and the zero-order light of slit #3 is arranged in the area of ​​the second-order light of slit #2.

[0117] Note that the distances of the three first-order lights do not have to be the same and can be set to be different. Therefore, the slit distances do not have to be the same and can be different.

[0118] As described above, according to the seventh example, since the zero-order light of a specific slit does not overlap with the first-order light of other slits even when the number of slits is three, accurate imaging data can be obtained in the imaging element 32. That is, more accurate diffraction data of the first-order light (with less noise) can be obtained. Note that the same can be achieved when the number of slits is four or more.

[0119] Next, Fig.17 is a graph for describing the limitation of the wavelength range to be observed in the present embodiment. The observation unit 1 may further include an optical filter that allows only light within a predetermined wavelength range among the obtained fluorescence to pass therethrough.

[0120] The actual fluorescence from the observation target may be diffused over a wide band. In this case, the wavelength range to be observed may be limited by using an optical filter or adjusting the sensitivity of the light receiving element. Specifically, for example, the following methods (A) to (C) are conceivable.

[0121] (A) A filter is placed in a portion of the emission spectrum to limit the wavelength.

[0122] (B) Using the sensitivity characteristics of the light receiving element to limit the wavelength to be observed.

[0123] (C) Combined methods (A) and (B).

[0124] By using these methods, for example, Fig.17 As shown in , the fluorescence in the area B1 can be limited and acquired as data in the area B2.

[0125] Next, Fig.18 : is a graph for describing data correction of second-order light in this embodiment. The horizontal axis represents wavelength, and the vertical axis represents light intensity. The processing unit 2 can estimate the amount of second-order light generated from the amount of first-order light with respect to the same light.

[0126] By acquiring the diffraction characteristics of the diffraction grating 35 in advance, for example, the amount of second-order light generated by slit #1 can be estimated based on the amount of first-order light from slit #1. That is, for example, in the case where the first-order light from slit #2 overlaps with the second-order light from slit #1, the signal of the first-order light from slit #2 can be acquired more accurately through data processing.

[0127] Specifically, although the efficiency of the second-order light is different from that of the first-order light, the waveform of the second-order light (which is the same waveform as that of the first-order light) appears in a wavelength region that is twice the wavelength of the first-order light. Therefore, by pre-evaluating the occurrence rate of the second-order light relative to the first-order light, it is possible to calculate how much second-order light is generated in the actual measurement. In the formula (13) provided below, the left side indicates the intensity of the second-order light, the first term on the right side indicates the intensity of the first-order light, and the second term on the right side indicates the occurrence rate of the second-order light.

[0128] I2(2λ)=I1(λ)×η2(λ) (13)

[0129] In this way, by calculating (estimating) the second-order light with high accuracy based on the measurement result of the first-order light, it is possible to perform data correction with high accuracy.

[0130] Next, Fig.19 is a graph for describing a specific example of data correction of second-order light in the present embodiment. An example of a method for evaluating the occurrence rate of second-order light will be described. For example, a filter that blocks light of a second-order light region to be evaluated is placed in a light source (such as a halogen lamp) having a wide emission wavelength characteristic. The light is introduced into the slits of a spectroscope respectively. Thus, the ratio of second-order light to first-order light can be evaluated, and the occurrence rate of second-order light can be evaluated.

[0131] exist Fig.19 In (a), reference numeral C1 represents the original light source spectrum, reference numeral C2 represents the filtered light source spectrum, and reference numeral C3 represents the second-order light occurrence rate evaluation area. Fig.19 In (b), reference symbol C4 represents the light source spectrum after filtering, and reference symbol C5 represents the second-order light of the light source after filtering. The ratio of reference symbol C5 to reference symbol C4 is the occurrence rate of the second-order light.

[0132] Furthermore, as an alternative to this method, for example, there is also a method of deriving the appearance rate of second-order light of each wavelength using a method of successively extracting monochromatic wavelengths from a high-brightness white light source.

[0133] Next, Fig. 20 is a graph for describing the data correction of the negative first-order light in the present embodiment. Although the efficiency of the negative first-order light is different from that of the first-order light, the waveform of the negative first-order light (which has the same waveform z as that of the first-order light) appears symmetrically with respect to the zero-order light. By pre-evaluating the occurrence rate of the negative first-order light and the first-order light, it is possible to calculate how much negative first-order light is generated in the actual measurement. In the equation (14) provided below, the left side indicates the intensity of the negative first-order light, the first term on the right side indicates the intensity of the first-order light, and the second term on the right side indicates the occurrence rate of the negative first-order light.

[0134] I -1 (λ)=I1(λ)×η -1 (λ) (14)

[0135] An example of a method for evaluating the appearance ratio of negative first-order light to first-order light will be described. When broadband light such as halogen lamp light is introduced into one slit, the spectrum of the dispersed first-order light appears in the data acquired from the imaging element 32. At this time, negative first-order light is generated at the same time. Thus, the appearance ratio of negative first-order light to first-order light can be evaluated. This is evaluated for each slit.

[0136] As an alternative to this method, there is also a method of deriving the appearance rate of negative first-order light of each wavelength using a method of continuously extracting monochromatic wavelengths from a high-intensity white light source.

[0137] Next, Fig.21is a diagram for describing a specific example of data correction in this embodiment. Fig.12 Data correction in case of D is the light intensity of region D and is the actual measured data. I1 is the intensity of the first-order light of slit #1. I2 is the intensity of the first-order light of slit #2. β -1 is the occurrence rate of the negative first-order light of slit #2 and is the pre-evaluated correction coefficient.

[0138] Moreover, I E is the light intensity of region E and is actual measurement data. α3 is the occurrence rate of the third-order light of slit #1 and is a correction coefficient estimated in advance. Then, I1 and I2 can be calculated by equations (15) and (16) provided below.

[0139] ID=I1+I2β -1 (15)

[0140] IE=I2+I1α3 (16)

[0141] Note that by applying Fig.18 The evaluation method for second-order light described in Fig. 20 The evaluation method described in β for negative first-order light is used to perform β -1 and the previous evaluation of α3.

[0142] In this way, the observation unit 1 of this embodiment is configured so that in the imaging element 32 , for multiple wavelength dispersed lights, the zero-order light of specific light and the first-order light of other lights do not overlap, so accurate imaging data can be obtained in the imaging element 32 .

[0143] For example, in the case of using a fluorescence microscope using multiple beams of excitation light and multiple slits in a conventional line scanning type fluorescence imaging device, there is a problem that the light sheets from the multiple slits are mixed with each other and accurate spectral data cannot be obtained. In contrast, by using the method according to the present embodiment, more accurate data with suppressed color mixing can be obtained.

[0144] Note that conventionally, only one line (or point) of light can be dispersed by one beam splitter. Therefore, in order to obtain spectral data of multiple lines (or points) at the same time, multiple beam splitters need to be configured. This is a complex system and imposes great restrictions on the physical layout. In observation systems such as microscopes, it is difficult to configure multiple beam splitters.

[0145] On the contrary, according to the present embodiment, a plurality of light beams (or points) can be split with high precision using one spectrometer (diffraction grating 35, imaging element 32, etc.). This allows the device to be simple, achieve a compact layout, and disperse multiple lines of light at the same time. In fluorescence observation, in the prior art, a line is scanned over the entire surface and the same number of shots as the number of excitation wavelengths are repeated, and a large amount of time is required. However, in the case where different excitation wavelengths are used for multiple lines, imaging can be completed in a single scan for excitation of multiple colors. That is, the observation time can be greatly shortened.

[0146] Furthermore, in this embodiment, if Fig. 9 As shown in the specific example of , the above effect can be achieved by configuring the observation unit 1 so that the zero-order light of the light passing through one slit is located between the zero-order light and the first-order light of the light passing through the other slits.

[0147] In addition, if Fig.10 As shown in the example of , the above-mentioned effect can be obtained by configuring the observation unit 1 so that the zero-order light of the light passing through one slit is located on the second-order light side relative to the first-order light of the light passing through the other slits.

[0148] In addition, regarding imaging data obtained from a plurality of wavelength-dispersed lights, respectively, by correcting imaging data obtained from other lights based on imaging data obtained from certain lights, the accuracy of the imaging data can be further improved.

[0149] Furthermore, by further using an optical filter that allows only light within a predetermined wavelength range in the obtained fluorescence to pass therethrough, the accuracy of the imaging data can be further improved.

[0150] In addition, when the observation object is a living body, it is effective that the wavelength band of the first-order light of at least one of the plurality of wavelength-dispersed lights is part or all of 400 to 800 nm.

[0151] Furthermore, by using a diffraction grating as a dispersion element, it is possible to easily design how wavelength dispersion occurs when light is incident on the imaging element 32 .

[0152] Furthermore, by using a blazed diffraction grating as the diffraction grating 35, a large amount of light of a specific order can be obtained, and even when there is a region where high-order light overlaps with first-order light, the influence can be suppressed to be small.

[0153] Furthermore, by setting the slit distance of the opening portion 31 to be equal to or smaller than the field diameter of the imaging data, imaging data based on a plurality of excitation lights can be accurately acquired.

[0154] Furthermore, by including the scanning mechanism 50 , two-dimensional data or three-dimensional data of the observation object can be acquired.

[0155] The fluorescence observation device 100 and a program for processing imaging data constitute a fluorescence observation system (microscope system). The program is a program for causing the information processing device to execute a correction process for processing imaging data.

[0156] This program is provided in a state of being stored in a recording medium such as a magnetic disk, an optical disk, a magneto-optical disk, or a flash memory, and is downloaded to an electronic computer or the like connected to the fluorescence observation apparatus 100 for use.

[0157] Furthermore, the program can be downloaded to an external electronic computer or the like via a network such as the Internet and used.

[0158] It should be noted that the present technology can also adopt the following configurations. (1)

[0160] A microscope device comprising:

[0161] an opening portion including a first slit and a second slit through which a plurality of beams of light from an observation object pass, the plurality of beams of light being generated by emitting a plurality of irradiation lights having different wavelengths to the observation object;

[0162] a dispersion element for performing wavelength dispersion on the plurality of light beams passing through the opening; and

[0163] The imaging element receives the multiple beams of light dispersed by the dispersion element.

[0164] The imaging element performs light reception so that, with respect to the wavelength-dispersed multiple beams of light, zero-order light of the light passing through the second slit and first-order light of the light passing through the first slit do not overlap each other. (2)

[0166] The microscope apparatus according to (1), further including an irradiation unit that irradiates the observation object with a plurality of irradiation lights having different wavelengths. (3)

[0168] The microscope apparatus according to (1) further includes a correction unit that processes imaging data received from the imaging element. (4)

[0170] The microscope apparatus according to (1), wherein the imaging element performs light reception so that zero-order light of the light passing through the second slit is located between zero-order light and first-order light of the light passing through the first slit. (5)

[0172] The microscope apparatus according to (1), wherein the imaging element performs light reception so that zero-order light of the light passing through the second slit is located on the second-order light side of the light passing through the first slit relative to first-order light of the light passing through the first slit. (6)

[0174] The microscope apparatus according to (3), wherein, for light information respectively obtained from the wavelength-dispersed plurality of lights, the correction unit corrects light information obtained from the light passing through the first slit based on light information obtained from the light passing through the second slit. (7)

[0176] The microscope apparatus according to (1) further includes a filter that allows only light within a predetermined wavelength range among the plurality of lights from the observation object to pass therethrough. (8)

[0178] The microscope apparatus according to (1), wherein a wavelength band of first-order light in at least one of the wavelength-dispersed plurality of light beams includes part or all of 400 nm to 800 nm. (9)

[0180] The microscope apparatus according to (1), wherein the dispersion element is a diffraction grating that causes wavelength dispersion due to coherence of light. (10)

[0182] The microscope apparatus according to (9), wherein the diffraction grating is a blazed diffraction grating in which the cross-sectional shape of the grooves is sawtooth-like. (11)

[0184] The microscope apparatus according to (1), wherein the imaging element receives a plurality of fluorescent lights obtained by irradiating the observation object with excitation light serving as a plurality of irradiation lights having different wavelengths. (12)

[0186] The microscope apparatus according to (1), wherein the slit distance of the opening portion is equal to or smaller than a field diameter of imaging data provided by the imaging element, which is defined by the objective lens and the imaging lens. (13)

[0188] The microscope device according to (3), further comprising:

[0189] The scanning mechanism changes the position of the object to be observed by the irradiated light over time,

[0190] By driving the scanning mechanism, the imaging element continuously images the wavelength-dispersed multiple beams in the scanning direction over time, and the correction unit processes the imaging data received from the imaging element to obtain two-dimensional data or three-dimensional data of the observation object. (14)

[0192] The microscope apparatus according to (2), wherein the irradiation unit emits the excitation light serving as the plurality of irradiation lights having different wavelengths in a linear shape. (15)

[0194] A beam splitter in a microscope device, comprising:

[0195] an opening portion including a first slit and a second slit through which a plurality of beams of light from an observation object pass, the plurality of beams of light being generated by emitting a plurality of irradiation lights having different wavelengths to the observation object;

[0196] a dispersion element for performing wavelength dispersion on the plurality of light beams passing through the opening; and

[0197] The imaging element receives the multiple beams of light dispersed by the dispersion element.

[0198] The imaging element performs light reception so that, with respect to the wavelength-dispersed multiple beams of light, zero-order light of the light passing through the second slit and first-order light of the light passing through the first slit do not overlap each other. (16)

[0200] A microscope system comprising: a microscope device according to any one of (1) to (14); and a program for processing imaging data imaged by the microscope device,

[0201] Here, the program is a program for causing an information processing device to execute correction processing for processing imaging data.

[0202] Although the embodiments of the present disclosure have been described above, the technical scope of the present disclosure is not limited to the above-described embodiments, and various modifications may be made without departing from the gist of the present disclosure.

[0203] For example, Figure 6 An example is shown in which the Offner type is used as the spectral imaging unit 30, and the curvature radius of the reflection surface on the outer side of the diffraction grating 35 is half the curvature radius of the reflection surface on the inner side of the reflection mirror 34, but the present disclosure is not limited thereto. Depending on the design, the ratio of their curvature radii may be different from 2:1.

[0204] Furthermore, the effects of the various embodiments described in this specification are merely illustrative and not limiting, and other effects may be provided.

[0205] Reference numerals list

[0206] 1 Observation unit

[0207] 2 Processing Units

[0208] 3 Display unit

[0209] 10 Excitation unit

[0210] 11 Collimating lens

[0211] 12 Laser Line Filters

[0212] 13 Dichroic Mirror

[0213] 14 Homogenizer

[0214] 15 Condenser lens

[0215] 16 Entrance slit

[0216] 20 Stage

[0217] 21 Storage Unit

[0218] 22 Data calibration unit

[0219] 23 Image forming unit

[0220] 30 Spectral Imaging Unit

[0221] 31 Opening

[0222] 32 Imaging elements

[0223] 33 Prism

[0224] 34 Reflector

[0225] 35 Diffraction grating

[0226] 40 Observation Optical System

[0227] 41 Condenser lens

[0228] 42 Dichroic Mirror

[0229] 43 Dichroic Mirror

[0230] 44 Objective lens

[0231] 45 Bandpass filter

[0232] 46 Condenser lens

[0233] 50 Scanning mechanism

[0234] 60 Focus on institutions

[0235] 70 Non-fluorescence observation unit

[0236] 80 control unit.

Claims

1. A microscope device comprising: an opening portion, comprising a first slit and a second slit, through which a plurality of beams of light from an observation object pass, the plurality of beams of light being generated by emitting a plurality of irradiation lights having different wavelengths to the observation object; a dispersion element for performing wavelength dispersion on the plurality of light beams passing through the opening; as well as an imaging element, receiving the multiple beams of light dispersed by the dispersion element, The imaging element performs light reception based on the adjustment of the distance between the first slit and the second slit so that for the multiple beams of light dispersed by wavelength, the zero-order light of the light passing through the second slit and the first-order light of the light passing through the first slit do not overlap with each other.

2. The microscope device according to claim 1, further comprising: An irradiation unit irradiates the observation object with a plurality of the irradiation lights having different wavelengths.

3. The microscope device according to claim 1, further comprising: A correction unit processes the imaging data received from the imaging element.

4. The microscope device according to claim 1, wherein: The imaging element performs light reception so that the zeroth-order light of the light passing through the second slit is located between the zeroth-order light of the light passing through the first slit and the first-order light.

5. The microscope device according to claim 1, wherein: The imaging element performs light reception such that the zeroth-order light of the light passing through the second slit is located on the second-order light side of the light passing through the first slit with respect to the first-order light of the light passing through the first slit.

6. The microscope device according to claim 3, wherein: The correction unit corrects the light information obtained from the light passing through the first slit based on the light information obtained from the light passing through the second slit, with respect to the light information respectively obtained from the plurality of wavelength-dispersed lights.

7. The microscope apparatus according to claim 1, further comprising: An optical filter that allows only light within a predetermined wavelength range among the plurality of light beams from the observation object to pass therethrough.

8. The microscope device according to claim 1, wherein: The wavelength band of the first-order light of at least one of the wavelength-dispersed plurality of light beams includes a part or all of 400 nm to 800 nm.

9. The microscope device according to claim 1, wherein: The dispersion element is a diffraction grating that causes wavelength dispersion due to the coherence of light.

10. The microscope device according to claim 9, wherein: The diffraction grating is a blazed diffraction grating in which the cross-sectional shape of the grooves is sawtooth-shaped.

11. The microscope device according to claim 1, wherein: The imaging element receives a plurality of fluorescent lights obtained by irradiating the observation object with excitation light serving as the plurality of irradiation lights having different wavelengths.

12. The microscope device according to claim 1, wherein: The slit distance of the opening is equal to or smaller than the field diameter of imaging data provided by the imaging element and defined by the objective lens and the imaging lens.

13. The microscope apparatus according to claim 3, further comprising: a scanning mechanism that changes the position of the observation object irradiated by the irradiation light over time, Wherein, by driving the scanning mechanism, the imaging element continuously images the multiple beams of wavelength-dispersed light in the scanning direction over time, and the correction unit processes the imaging data received from the imaging element to obtain two-dimensional data or three-dimensional data of the observed object.

14. The microscope device according to claim 2, wherein: The irradiation unit emits excitation light serving as the plurality of irradiation lights having different wavelengths in a linear shape.

15. A spectrometer in a microscope device, the spectrometer comprising: an opening portion, comprising a first slit and a second slit, through which a plurality of beams of light from an observation object pass, the plurality of beams of light being generated by emitting a plurality of irradiation lights having different wavelengths to the observation object; a dispersion element for performing wavelength dispersion on the plurality of light beams passing through the opening; as well as an imaging element, receiving the multiple beams of light dispersed by the dispersion element, The imaging element performs light reception based on the adjustment of the distance between the first slit and the second slit so that for the multiple beams of light dispersed by wavelength, the zero-order light of the light passing through the second slit and the first-order light of the light passing through the first slit do not overlap with each other.

16. A microscope system comprising: The microscope device according to any one of claims 1 to 14; and a computer-readable storage medium having stored thereon a program for processing imaging data imaged by the microscope device, When the program is executed by an information processing device, the program causes the information processing device to perform correction processing for processing the imaging data.

Images